RESUMO
Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has shown that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyze lysosome morphology, positioning, motility and function. We highlight the principles behind these methods, the methodological strategies and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground-breaking discoveries on an organelle that continues to surprise and excite cell biologists.
Assuntos
Lisossomos , Redes e Vias Metabólicas , Lisossomos/metabolismo , Transdução de SinaisRESUMO
Mucopolysaccharidosis type IIIA (MPS-IIIA) is an autosomal recessive disorder caused by mutations in SGSH involved in the degradation of heparan sulfate. MPS-IIIA presents severe neurological symptoms such as progressive developmental delay and cognitive decline, for which there is currently no treatment. Brain targeting represents the main challenge for therapeutics to treat MPS-IIIA, and the development of small-molecule-based treatments able to reach the CNS could be a relevant advance for therapy. Using cell-based high content imaging to survey clinically approved drugs in MPS-IIIA cells, we identified fluoxetine, a selective serotonin reuptake inhibitor. Fluoxetine increases lysosomal and autophagic functions via TFEB activation through a RagC-dependent mechanism. Mechanistically, fluoxetine increases lysosomal exocytosis in mouse embryonic fibroblasts from MPS-IIIA mice, suggesting that this process may be responsible for heparan sulfate clearance. In vivo, fluoxetine ameliorates somatic and brain pathology in a mouse model of MPS-IIIA by decreasing the accumulation of glycosaminoglycans and aggregated autophagic substrates, reducing inflammation, and slowing down cognitive deterioration. We repurposed fluoxetine for potential therapeutics to treat human MPS-IIIA disease.
Assuntos
Mucopolissacaridose III , Animais , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fluoxetina/farmacologia , Fluoxetina/uso terapêutico , Heparitina Sulfato/metabolismo , Hidrolases/genética , Camundongos , Mucopolissacaridose III/tratamento farmacológico , Mucopolissacaridose III/genéticaRESUMO
According to WHO, breast cancer incidence is increasing so that the search for novel chemotherapeutic options is nowadays an essential requirement to fight neoplasm subtypes. By exploring new effective metal-based chemotherapeutic strategies, many ruthenium complexes have been recently proposed as antitumour drugs, showing ability to impact on diverse cellular targets. In the framework of different molecular pathways leading to cell death in human models of breast cancer, here we demonstrate autophagy involvement behind the antiproliferative action of a ruthenium(III)-complex incorporated into a cationic nanosystem (HoThyRu/DOTAP), proved to be hitherto one of the most effective within the suite of nucleolipidic formulations we have developed for the in vivo transport of anticancer ruthenium(III)-based drugs. Indeed, evidences are implicating autophagy in both cancer development and therapy, and anticancer interventions endowed with the ability to trigger this biological response are currently considered attractive oncotherapeutic approaches. Moreover, crosstalk between apoptosis and autophagy, regulated by finely tuned metallo-chemotherapeutics, may provide novel opportunities for future improvement of cancer treatment. Following this line, our in vitro and in vivo preclinical investigations suggest that an original strategy based on suitable formulations of ruthenium(III)-complexes, inducing sustained cell death, could open new opportunities for breast cancer treatment, including the highly aggressive triple-negative subtype.
Assuntos
Antineoplásicos/administração & dosagem , Proteínas Relacionadas à Autofagia/metabolismo , Neoplasias da Mama/metabolismo , Complexos de Coordenação/administração & dosagem , Ácidos Graxos Monoinsaturados/química , Compostos de Amônio Quaternário/química , Rutênio/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Autofagia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos , Estrutura Molecular , Nanopartículas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Bisphenol A (BPA) is a chemical used in numerous industrial applications. Due to its well ascertained toxicity as endocrine disruptor, industries have started to replace it with other bisphenols whose alleged greater safety is scarcely supported by literature studies. In this study, the toxicity of seven BPA analogues was evaluated using both in silico and in vitro techniques, as compared to BPA toxicity. Furthermore, their affinity indexes for phospholipids (i.e. phospholipophilicity) were determined by immobilized artificial membrane liquid chromatography (IAM-LC) and possible relationships with in vitro toxic activity were also investigated. The results on four different cell cultures yielded similar ranking of toxicity for the bisphenols considered, with IC50 values confirming their poor acute toxicity. As compared to BPA, bisphenol AF, bisphenol B, bisphenol M, and bisphenol A diglycidyl ether resulted more toxic, while bisphenol S, bisphenol F and bisphenol E were found as the less toxic congeners. These results are partly consistent with the scale of phospholipid affinity showing that toxicity increases at increasing membrane affinity. Therefore, phospholipophilicity determination can be assumed as a useful preliminary tool to select less toxic congeners to surrogate BPA in industrial applications.
Assuntos
Compostos Benzidrílicos/toxicidade , Fenóis/toxicidade , Animais , Linhagem Celular , Cromatografia Líquida , Disruptores Endócrinos/análise , Compostos de Epóxi/toxicidade , Humanos , Membranas Artificiais , Camundongos , Fosfolipídeos/metabolismo , Ratos , Sulfonas/toxicidadeRESUMO
In the search for more efficient anticancer treatments, Ru(III) complexes have attracted much interest among metal-based candidate drugs, showing marked antitumor and antimetastatic activity associated with lower systemic toxicity. Remarkable examples are the Ru(III) complexes NAMI-A and KP1019, which have reached advanced clinical evaluation. In order to improve the in vivo stability of Ru(III)-based drugs, as well as their cellular uptake and effectiveness, a new approach has been proposed by our research group, based on the incorporation of the active, NAMI-A-like Ru(III) complex into highly functionalized nucleolipidic structures, i.e., hybrid molecules containing a nucleoside or nucleotide central core derivatized with a lipid chain, ensuring both efficient protection against extracellular degradation and high cellular internalization of the metal. Aiming at expanding the chemical diversity of available amphiphilic Ru(III) complexes, we here selected a trifunctional α-amino acid to replace the nucleosidic core of previously prepared nucleolipid-based Ru(III) complexes. The amino acidic scaffold, linked to the Ru(III) complex, is decorated with both hydrophilic and lipophilic moieties, conferring high propensity to form stable aggregates in water, which is required to obtain a suitable nanocarrier for the drug delivery. Following this approach, a novel compound, indicated here as compound I, was successfully prepared and characterized, then studied in coformulation with the biocompatible cationic lipid 1,2-dioleyl-3-trimethylammoniumpropane chloride (DOTAP) by dynamic light scattering (DLS), small angle neutron scattering (SANS), and UV-vis analysis. Evaluated in vitro on a panel of human and nonhuman cell lines, it showed good antiproliferative activity on cancer cells, with IC50 values in the µM range, and no relevant cytotoxicity on the healthy cells used as control.
RESUMO
Looking for new metal-based anticancer treatments, in recent years many ruthenium complexes have been proposed as effective and safe potential drugs. In this context we have recently developed a novel approach for the in vivo delivery of Ru(III) complexes, preparing stable ruthenium-based nucleolipidic nanoaggregates endowed with significant antiproliferative activity. Herein we describe the cellular response to our ruthenium-containing formulations in selected models of human breast cancer. By in vitro bioscreens in the context of preclinical studies, we have focused on their ability to inhibit breast cancer cell proliferation by the activation of the intrinsic apoptotic pathway, possibly via mitochondrial perturbations involving Bcl-2 family members and predisposing to programmed cell death. In addition, the most efficient ruthenium-containing cationic nanoaggregates we have hitherto developed are able to elicit both extrinsic and intrinsic apoptosis, as well as autophagy. To limit chemoresistance and counteract uncontrolled proliferation, multiple cell death pathways activation by metal-based chemotherapeutics is a challenging, yet very promising strategy for targeted therapy development in aggressive cancer diseases, such as triple-negative breast cancer with limited treatment options. These outcomes provide valuable, original knowledge on ruthenium-based candidate drugs and new insights for future optimized cancer treatment protocols.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Proliferação de Células/efeitos dos fármacos , Nanocompostos/química , Compostos Organometálicos/farmacologia , Rutênio/química , Apoptose/efeitos dos fármacos , Humanos , Células MCF-7 , Compostos Organometálicos/químicaRESUMO
Nuclear Factor-κB (NF-κB) is a transcription factor regulating several genes involved in important physiological and pathological processes. NF-κB has been found constitutively activated in many inflammatory/immune diseases. In addition, a positive correlation between persistent activation of NF-κB and tumor promotion has been demonstrated. Since the IKK (IκB kinase) activation is an indispensable component of all pro-inflammatory signaling pathways leading to NF-κB activation, considerable efforts have been done in order to develop novel anti-inflammatory therapeutics targeting IKK. Association of the IKK complex relies on critical interactions between the C-terminus NBD (NEMO binding domain) of the catalytic subunits IKKα and IKKß, and the regulatory subunit NEMO (NF-κB Essential Modulator). Thus, this IKK/NEMO interacting region provides an attractive target to prevent the IKK complex formation and NF-κB activation. In this regard, we have identified non-peptide small molecule disruptors of IKKß/NEMO complex through a structure-based virtual screening (SBVS) of the NCI chemical library. Phenothiazine 22 and its close analogues (22.2, 22.4 and 22.10) were able to reduce nitrite production and iNOS mRNA expression in J774 murine macrophages stimulated with LPS for 24h. These effects were associated with a reduced NF-κB/DNA binding activity as well as a decreased expression of phosphorylated IKKß, IκBα and NF-κB/p65 in these cells. These observations suggest that compound 22 and its three structural analogues by inhibiting IKKß/NEMO association mediate the blockage of NF-κB signaling pathway and may prove effective in treatment of diseases in which the IKK/NF-κB pathway is dysregulated.
Assuntos
Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Descoberta de Drogas , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Camundongos , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/químicaRESUMO
L-cysteine is currently recognized as a conditionally essential sulphur amino acid. Besides contributing to many biological pathways, cysteine is a key component of the keratin protein by its ability to form disulfide bridges that confer strength and rigidity to the protein. In addition to cysteine, iron represents another critical factor in regulating keratins expression in epidermal tissues, as well as in hair follicle growth and maturation. By focusing on human keratinocytes, the aim of this study was to evaluate the effect of cysteine supplementation as nutraceutical on keratin biosynthesis, as well as to get an insight on the interplay of cysteine availability and cellular iron status in regulating keratins expression in vitro. Herein we demonstrate that cysteine promotes a significant up-regulation of keratins expression as a result of de novo protein synthesis, while the lack of iron impairs keratin expression. Interestingly, cysteine supplementation counteracts the adverse effect of iron deficiency on cellular keratin expression. This effect was likely mediated by the up-regulation of transferrin receptor and ferritin, the main cellular proteins involved in iron homeostasis, at last affecting the labile iron pool. In this manner, cysteine may also enhance the metabolic iron availability for DNA synthesis without creating a detrimental condition of iron overload. To the best of our knowledge, this is one of the first study in an in vitro keratinocyte model providing evidence that cysteine and iron cooperate for keratins expression, indicative of their central role in maintaining healthy epithelia.
Assuntos
Cisteína/farmacologia , Ferro/metabolismo , Queratinócitos/metabolismo , Queratinas/biossíntese , Linhagem Celular , Homeostase , Humanos , Queratinócitos/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Regulação para CimaRESUMO
Signaling through mammalian target of rapamycin (mTOR) has been shown to play a central role in the regulation of skeletal muscle growth induced by a wide range of stimuli either mechanical or metabolic, such as growth factors and amino acids. Here, we demonstrate that mTOR and its downstream target, the ribosomal S6 kinase (p70(S6K)), are activated in L6 myocytes by a short-term glucose deprivation. Such response is specific of skeletal muscle and is likely responsible for the increased rate of protein synthesis and expression of the muscle-specific proteins during recovery from glucose deprivation. Nitric oxide and phosphatidylinositol-3-kinase (PI3K) are upstream positive regulators of mTOR since their pharmacological inhibition prevents the activation of p70(S6K) in response to glucose deprivation. We therefore propose a model of response to a brief period of glucose deprivation that may occur in skeletal muscle cells during resistance exercise and that may lead to protein accretion when blood flow recovers and all nutrients are again available.
Assuntos
Glucose/deficiência , Mioblastos Esqueléticos/metabolismo , Biossíntese de Proteínas , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Células CACO-2 , Glucose/metabolismo , Células HEK293 , Humanos , Contração Muscular , Mioblastos Esqueléticos/fisiologia , Óxido Nítrico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Proteínas Quinases S6 Ribossômicas/metabolismoRESUMO
Aiming for novel tools for anticancer therapies, a ruthenium complex, covalently linked to a cholesterol-containing nucleolipid and stabilized by co-aggregation with a biocompatible lipid, is here presented. The amphiphilic ruthenium complex, named ToThyCholRu, is intrinsically negatively charged and has been inserted into liposomes formed by the cationic 1,2-dioleyl-3-trimethylammoniumpropane chloride (DOTAP) to hinder the degradation kinetics typically observed for known ruthenium-based antineoplastic agents. The here described nanovectors contain up to 30% in moles of the ruthenium complex and are stable for several weeks. This drug delivery system has been characterized using dynamic light scattering (DLS), small angle neutron scattering (SANS), neutron reflectivity (NR) and electron paramagnetic resonance (EPR) techniques. Fluorescence microscopy, following the incorporation of rhodamine-B within the ruthenium-loaded liposomes, showed fast cellular uptake in human carcinoma cells, with a strong fluorescence accumulation within the cells. The in vitro bioactivity profile revealed an important antiproliferative activity and, most remarkably, the highest ability in ruthenium vectorization measured so far. Cellular morphological changes and DNA fragmentation provided evidence of an apoptosis-inducing activity, in line with several in vitro studies supporting apoptotic events as the main cause for the anticancer properties of ruthenium derivatives. Overall, these data highlighted the crucial role played by the cellular uptake properties in determining the anticancer efficacy of ruthenium-based drugs, showing DOTAP as a very efficient nanocarrier for their stabilization in aqueous media and transport in cells. In vitro bioscreens have shown the high antiproliferative activity of ToThyCholRu-DOTAP liposomes against specific human adenocarcinoma cell types. Furthermore, these formulations have proved to be over 20-fold more effective against MCF-7 and WiDr adenocarcinoma cells with respect to the nude ruthenium complex AziRu we have previously described.
RESUMO
Degradation of nucleic acids in biological environments is the major drawback of the therapeutic use of aptamers. Among the approaches used to circumvent this negative aspect, the introduction of 3'-3' inversion of polarity sites at the sequence 3'-end has successfully been proposed. However, the introduction of inversion of polarity at the ends of the sequence has never been exploited for G-quadruplex forming aptamers. In this communication we describe CD, UV, electrophoretic and biochemical investigations concerning thrombin binding aptamer analogues containing one or two inversions of polarity sites at the oligonucleotide ends. Data indicate that, in some cases, this straightforward chemical modification is able to improve, at the same time, the thermal stability, affinity to thrombin and nuclease resistance in biological environments, thus suggesting its general application as a post-SELEX modification also for other therapeutically promising aptamers adopting G-quadruplex structures.
Assuntos
Oligonucleotídeos/química , Trombina/química , Sítios de Ligação , Quadruplex G , Trombina/análogos & derivadosRESUMO
In order to expand the potential applications of G-quadruplex structures, we explored the ability of heterochiral oligodeoxynucleotides based on the thrombin-binding aptamer (TBA) sequence to fold into similar complexes, with particular focus on their resistance in biological environments. A combination of CD and NMR techniques was used. Similarly to TBA, the ODN ggTTggtgtggTTgg (lower case letters indicate L residues) is able to fold into a chair-like antiparallel G-quadruplex structure, but has a slightly higher thermal stability. The discovery that heterochiral ODNs are able to form stable G-quadruplex structures opens up new possibilities for their development in several fields, as aptamers, sensors and, as recently shown, as catalysts for enantioselective reactions.